The present invention relates to cancer therapy. More specifically, the present invention relates to the inhibition of the proliferation of cancer cells using modified antisense oligonucleotides complementary to nucleic acid encoding the protein kinase A RIxcex1 subunit.
The development of effective cancer therapies has been a major focus of biomedical research. Surgical procedures have been developed and used to treat patients whose tumors are confined to particular anatomical sites. However, at present, only about 25% of patients have tumors that are truly confined and amenable to surgical treatment alone (Slapak et al. in Harrison""s Principles of Internal Medicine (Isselbacher et al., eds.) McGraw-Hill, Inc., NY (1994) pp. 1826-1850). Radiation therapy, like surgery, is a local modality whose usefulness in the treatment of cancer depends to a large extent on the inherent radiosensitivity of the tumor and its adjacent normal tissues. However, radiation therapy is associated with both acute toxicity and long term sequelae. Furthermore, radiation therapy is known to be mutagenic, carcinogenic, and teratogenic (Slapak et al., ibid.).
Systemic chemotherapy alone or in combination with surgery and/or radiation therapy is currently the primary treatment available for disseminated malignancies. However, conventional chemotherapeutic agents which either block enzymatic pathways or randomly interact with DNA irrespective of the cell phenotype, lack specificity for killing neoplastic cells. Thus, systemic toxicity often results from standard cytotoxic chemotherapy. More recently, the development of agents that block replication, transcription, or translation in transformed cells, and at the same time defeat the ability of cells to become resistant, has been the goal of many approaches to chemotherapy.
One strategy is to down regulate the expression of a gene associated with the neoplastic phenotype in a cell. A technique for turning off a single activated gene is the use of antisense oligodeoxynucleotides and their analogues for inhibition of gene expression (Zamecnik et al. (1978) Proc. Natl. Acad. Sci. (USA) 75:280-284). An antisense oligonucleotide targeted at a gene involved in the neoplastic cell growth should specifically interfere only with the expression of that gene, resulting in arrest of cancer cell growth. The ability to specifically block or down-regulate expression of such genes provides a powerful tool to explore the molecular basis of normal growth regulation, as well as the opportunity for therapeutic intervention (see, e.g., Cho-Chung (1993) Curr. Opin. Thera. Patents 3:1737-1750). The identification of genes that confer a growth advantage to neoplastic cells as well as other genes causally related to cancer and the understanding of the genetic mechanism(s) responsible for their activation makes the antisense approach to cancer treatment possible.
One such gene encodes the RIxcex1 subunit of cyclic AMP (cAMP)-dependent protein kinase A (PKA) (Krebs (1972) Curr. Topics Cell. Regul. 5:99-133). Protein kinase is bound by cAMP, which is thought to have a role in the control of cell proliferation and differentiation (see, e.g., Cho-Chung (1980) J. Cyclic Nucleotide Res. 6:163-167). There are two types of PKA, type I (PKA-I) and type II (PKA-II), both of which share a common C subunit but each containing distinct R subunits, RI and RII, respectively (Beebe et al. in The Enzymes: Control by Phosphorylation, 17(A):43-111 (Academic, New York, 1986). The R subunit isoforms differ in tissue distribution (Øyen et al. (1988) FEBS Lett. 229:391-394; Clegg et al. (1988) Proc. Natl. Acad. Sci. (USA) 85:3703-3707) and in biochemical properties (Beebe et al. in The Enzymes: Control by Phosphorylation, 17(A):43-111 (Academic Press, NY, 1986); Cadd et al. (1990) J. Biol. Chem. 265:19502-19506). The two general isoforms of the R subunit also differ in their subcellular localization: RI is found throughout the cytoplasm; whereas RII localizes to nuclei, nucleoli, Golgi apparatus and the microtubule-organizing center (see, e.g., Lohmann in Advances in Cyclic Nucleotide and Protein Phosphorylation Research, 18:63-117 (Raven, New York, 1984; and Nigg et al. (1985) Cell 41:1039-1051).
An increase in the level of RIxcex1 expression has been demonstrated in human cancer cell lines and in primary tumors, as compared with normal counterparts, in cells after transformation with the Ki-ras oncogene or transforming growth factor-xcex1, and upon stimulation of cell growth with granulocyte-macrophage colony-stimulating factor (GM-CSF) or phorbol esters (Lohmann in Advances in Cyclic Nucleotide and Protein Phosphorylation Research, 18:63-117 (Raven, New York, 1984); and Cho-Chung (1990) Cancer Res. 50:7093-7100). Conversely, a decrease in the expression of RIxcex1 has been correlated with growth inhibition induced by site-selective cAMP analogs in a broad spectrum of human cancer cell lines (Cho-Chung (1990) Cancer Res. 50:7093-7100). It has also been determined that the expression of RI/PKA-I and RII/PKA-II has an inverse relationship during ontogenic development and cell differentiation (Lohmann in Advances in Cyclic Nucleotide and Protein Phosphorylation Research, Vol. 18, 63-117 (Raven, New York, 1984); Cho-Chung (1990) Cancer Res. 50:7093-7100). The RIxcex1 subunit of PKA has thus been hypothesized to be an ontogenic growth-inducing protein whose constitutive expression disrupts normal ontogenic processes, resulting in a pathogenic outgrowth, such as malignancy (Nesterova et al. (1995) Nature Medicine 1:528-533).
Antisense oligonucleotides directed to the RIxcex1 gene have been prepared. U.S. Pat. No. 5,271,941 describes phosphodiester-linked antisense oligonucleotides complementary to a region of the first 100 N-terminal amino acids of RIxcex1 which inhibit the expression of RIxcex1 in leukemia cells in vitro. In addition, antisense phosphorothioate oligodeoxynucleotides corresponding to the N-terminal 8-13 codons of the RIxcex1 gene was found to reduce in vivo tumor growth in nude mice (Nesterova et al. (1995) Nature Med. 1:528-533).
Unfortunately, problems have been encountered with the use of phosphodiester-linked (PO) oligonucleotides and some phosphorothioate-linked (PS) oligonucleotides. It is known that nucleases in the serum readily degrade PO oligonucleotides. Replacement of the phosphodiester internucleotide linkages with phosphorothioate internucleotide linkages has been shown to stabilize oligonucleotides in cells, cell extracts, serum, and other nuclease-containing solutions (see, e.g., Bacon et al. (1990) Biochem. Biophys. Meth. 20:259) as well as in vivo (Iversen (1993) Antisense Research and Application (Crooke, ed) CRC Press, 461). However, some PS oligonucleotides have been found to exhibit an immunostimulatory response, which in certain cases may be undesirable. For example, Galbraith et al. (Antisense Res. and Dev. (1994) 4:201-206) disclose complement activation by some PS oligonucleotides. Henry et al. (Pharm. Res. (1994) 11: PPDM8082) disclose that some PS oligonucleotides may potentially interfere with blood clotting.
There is, therefore, a need for modified oligonucleotides directed to cancer-related genes that retain gene expression inhibition properties while producing fewer side effects than conventional oligonucleotides.
The present invention relates to modified oligonucleotides useful for studies of gene expression and for the antisense therapeutic approach. The invention provides modified oligonucleotides that down-regulate the expression of the RIxcex1 gene while producing fewer side effects than conventional oligonucleotides. In particular, the invention provides modified oligonucleotides that demonstrate reduced mitogenicity, reduced activation of complement and reduced antithrombotic properties, relative to conventional oligonucleotides.
It is also known that some PS oligonucleotides cause an immunostimulatory response in subjects to whom they have been administered, which may be undesirable in some cases.
It is known that exclusively phosphodiester- or exclusively phosphorothioate-linked oligonucleotides directed to the first 100 nucleotides of the RIxcex1 nucleic acid inhibit cell proliferation.
It has now been discovered that modified oligonucleotides complementary to the protein kinase A RIxcex1 subunit gene inhibit the growth of tumors in vivo. With at least the activity of a comparable PO- or PS-linked oligonucleotide with fewer side effects.
This finding has been exploited to produce the present invention, which in a first aspect, includes synthetic hybrid, inverted hybrid, and inverted chimeric oligonucleotides and compositions of matter for specifically down-regulating protein kinase A subunit RIxcex1 gene expression with reduced side effects. Such inhibition of gene expression is useful as an alternative to mutant analysis for determining the biological function and role of protein kinase A-related genes in cell proliferation and tumor growth. Such inhibition of RIxcex1 gene expression can also be used to therapeutically treat diseases and disorders that are caused by the over-expression or inappropriate expression of the gene.
As used herein, the term xe2x80x9csynthetic oligonucleotidexe2x80x9d includes chemically synthesized polymers of three up to 50, preferably from about 15 to about 30, and most preferably, 18 ribonucleotide and/or deoxyribonucleotide monomers connected together or linked by at least one, and preferably more than one, 5xe2x80x2 to 3xe2x80x2 internucleotide linkage.
For purposes of the invention, the term xe2x80x9coligonucleotide sequence that is complementary to a genomic region or an RNA molecule transcribed therefromxe2x80x9d is intended to mean an oligonucleotide that binds to the nucleic acid sequence under physiological conditions, e.g., by Watson-Crick base pairing (interaction between oligonucleotide and single-stranded nucleic acid) or by Hoogsteen base pairing (interaction between oligonucleotide and double-stranded nucleic acid) or by any other means including in the case of a oligonucleotide binding to RNA, causing pseudoknot formation. Binding by Watson-Crick or Hoogsteen base pairing under physiological conditions is measured as a practical matter by observing interference with the function of the nucleic acid sequence.
In one preferred embodiment according to this aspect of the invention, the oligonucleotide is a core region hybrid oligonucleotide comprising a region of at least two deoxyribonucleotides, flanked by 5xe2x80x2 and 3xe2x80x2 ribonucleotide regions, each having at least four ribonucleotides. A hybrid oligonucleotide having the sequence set forth in the Sequence Listing as SEQ ID NO:4 is one particular embodiment. In some embodiments, each of the 3xe2x80x2 and 5xe2x80x2 flanking ribonucleotide regions of an oligonucleotide of the invention comprises at least four contiguous, 2xe2x80x2-O-substituted ribonucleotides.
For purposes of the invention, the term xe2x80x9c2xe2x80x2-O-substitutedxe2x80x9d means substitution of the 2xe2x80x2 position of the pentose moiety with an -O-lower alkyl group containing 1-6 saturated or unsaturated carbon atoms, or with an -O-aryl or allyl group having 2-6 carbon atoms, wherein such alkyl, aryl or allyl group may be unsubstituted or may be substituted, e.g., with halo, hydroxy, trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxyl, carbalkoxyl, or amino groups; or with a hydroxy, an amino or a halo group, but not with a 2xe2x80x2-H group.
In some embodiments, each of the 3xe2x80x2 and 5xe2x80x2 flanking ribonucleotide regions of an oligonucleotide of the invention comprises at least one 2xe2x80x2-O-alkyl substituted ribonucleotide. In one preferred embodiment, the 2xe2x80x2-O-alkyl-substituted nucleotide is a 2xe2x80x2-O-methyl ribonucleotide. In other preferred embodiments, the 3xe2x80x2 and 5xe2x80x2 flanking ribonucleotide regions of an oligonucleotide of the invention comprises at least four 2xe2x80x2-O-methyl ribonucleotides. In preferred embodiments, the ribonucleotides and deoxyribonucleotides of the hybrid oligonucleotide are linked by phosphorothioate internucleotide linkages. In particular embodiments, this phosphorothioate region or regions have from about four to about 18 nucleosides joined to each other by 5xe2x80x2 to 3xe2x80x2 phosphorothioate linkages, and preferably from about 5 to about 18 such phosphorothioate-linked nucleosides. The phosphorothioate linkages may be mixed Rp and Sp enantiomers, or they may be stereoregular or substantially stereoregular in either Rp or Sp form (see Iyer et al. (1995) Tetrahedron Asymmetry 6:1051-1054).
In another preferred embodiment according to this aspect of the invention, the oligonucleotide is an inverted hybrid oligonucleotide comprising a region of at least four ribonucleotides flanked by 3xe2x80x2 and 5xe2x80x2 deoxyribonucleotide regions of at least two deoxyribonucleotides. The structure of this oligonucleotide is xe2x80x9cinvertedxe2x80x9d relative to traditional hybrid oligonucleotides. In some embodiments, the 2xe2x80x2-O-substituted RNA region has from about four to about ten 2xe2x80x2-O-substituted nucleosides joined to each other by 5xe2x80x2 to 3xe2x80x2 internucleoside linkages, and most preferably from about four to about six such 2xe2x80x2-O-substituted nucleosides. In some embodiments, the oligonucleotides of the invention have a ribonucleotide region that comprises at least five contiguous ribonucleotides. In one particularly preferred embodiment, the overall size of the inverted hybrid oligonucleotide is 18. In preferred embodiments, the 2xe2x80x2-O-substituted ribonucleosides are linked to each other through a 5xe2x80x2 to 3xe2x80x2 phosphorothioate, phosphorodithioate, phosphotriester, or phosphodiester linkages. The phosphorothioate 3xe2x80x2 or 5xe2x80x2 flanking region (or regions) has from about four to about 18 nucleosides joined to each other by 5xe2x80x2 to 3xe2x80x2 phosphorothioate linkages, and preferably from about 5 to about 18 such phosphorothioate-linked nucleosides. In preferred embodiments, the phosphorothioate regions will have at least 5 phosphorothioate-linked nucleosides. One specific embodiment is an oligonucleotide having substantially the nucleotide sequence set forth in the Sequence Listing as SEQ ID NO:6. In preferred embodiments of this aspect of the invention, the ribonucleotide region comprises 2xe2x80x2-O-substituted ribonucleotides, such as 2xe2x80x2-O-alkyl substituted ribonucleotides. One particularly preferred embodiment is an inverted hybrid oligonucleotide whose ribonucleotide region comprises at least one 2xe2x80x2-O-methyl ribonucleotide.
In some embodiments, all of the nucleotides in the inverted hybrid oligonucleotide are linked by phosphorothioate internucleotide linkages. In particular embodiments, the deoxyribonucleotide flanking region or regions has from about four to about 18 nucleosides joined to each other by 5xe2x80x2 to 3xe2x80x2 phosphorothioate linkages, and preferably from about 5 to about 18 such phosphorothioate-linked nucleosides. In some embodiments, the deoxyribonucleotide 3xe2x80x2 and 5xe2x80x2 flanking regions of the inverted hybrid oligonucleotides of the invention have about 5 phosphorothioate-linked nucleosides. The phosphorothioate linkages may be mixed Rp and Sp enantiomers, or they may be stereoregular or substantially stereoregular in either Rp or Sp form (see Iyer et al. (1995) Tetrahedron Asymmetry 6:1051-1054)
Another embodiment is a composition of matter for inhibiting the expression of protein kinase A subunit RIxcex1 with reduced side effects, the composition comprising an inverted hybrid oligonucleotide according to the invention.
Yet another preferred embodiment according to this aspect of the invention is an inverted chimeric oligonucleotide comprising an oligonucleotide nonionic region of at least four nucleotides flanked by one or more, and preferably two oligonucleotide phosphorothioate regions. Such a chimeric oligonucleotide has a structure that is xe2x80x9cinvertedxe2x80x9d relative to traditional chimeric oligonucleotides. In one particular embodiment, an inverted chimeric oligonucleotide of the invention has substantially the nucleotide sequence set forth in the Sequence Listing as SEQ ID NO:1. In preferred embodiments, the oligonucleotide nonionic region comprises about four to about 12 nucleotides joined to each other by 5xe2x80x2 to 3xe2x80x2 nonionic linkages. In some embodiments, the nonionic region contains alkylphosphonate and/or phosphoramidate and/or phosphotriester internucleoside linkages. In one particular embodiment, the oligonucleotide nonionic region comprises six nucleotides. In some preferred embodiments, the oligonucleotide has a nonionic region having from about six to about eight methylphosphonate-linked nucleosides, flanked on either side by phosphorothioate regions, each having from about six to about ten phosphorothioate-linked nucleosides. In preferred embodiments, the flanking region or regions are phosphorothioate nucleotides. In some embodiments, the flanking region or regions have from about four to about 24 nucleosides joined to each other by 5xe2x80x2 to 3xe2x80x2 phosphorothioate linkages, and preferably from about six to about 16 such phosphorothioate-linked nucleosides. In preferred embodiments, the phosphorothioate regions have from about five to about 15 phosphorothioate-linked nucleosides. The phosphorothioate linkages may be mixed Rp and Sp enantiomers, or they may be stereoregular or substantially stereoregular in either Rp or Sp form (see Iyer et al. (1995) Tetrahedron Asymmetry 6:1051-1054).
Another embodiment of this aspect of the invention is a composition of matter for inhibiting the expression of protein kinase A subunit RIxcex1 with reduced side effects, the composition comprising an inverted chimeric oligonucleotide according to the invention.
Another aspect of the invention is a method of inhibiting the proliferation of cancer cells in vitro. In this method, an oligonucleotide of the invention is administered to the cells.
Yet another aspect is a therapeutic composition comprising an oligonucleotide of the invention in a pharmaceutically acceptable carrier.
A method of treating cancer in an afflicted subject with reduced side effects is another aspect of the invention. This method comprises administering a therapeutic composition of the invention to the subject in which the protein kinase A subunit RIxcex1 gene is being over-expressed.
Those skilled in the art will recognize that the elements of these preferred embodiments can be combined and the inventor does contemplate such combination. For example, 2xe2x80x2-O-substituted ribonucleotide regions may well include from one to all nonionic internucleoside linkages. Alternatively, nonionic regions may have from one to all 2xe2x80x2-O-substituted ribonucleotides. Moreover, oligonucleotides according to the invention may contain combinations of one or more 2xe2x80x2-O-substituted ribonucleotide region and one or more nonionic region, either or both being flanked by phosphorothioate regions. (See Nucleosides and Nucleotides 14:1031-1035 (1995) for relevant synthetic techniques.)